期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 287, 期 12, 页码 9213-9221出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M111.314286
关键词
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资金
- United States-Israel Binational Science Foundation, Jerusalem, Israel
- Israeli Centers of Research Excellence (I-CORE) [Center 152/11]
- Alternative Energy Research Initiative Bioenergy Consortium
- Weizmann Institute of Science Alternative Energy Research Initiative
- Israel Science Foundation [966/09, 159/07, 24/11]
beta-Xylosidases are hemicellulases that hydrolyze short xylo-oligosaccharides into xylose units, thus complementing endoxylanase degradation of the hemicellulose component of lignocellulosic substrates. Here, we describe the cloning, characterization, and kinetic analysis of a glycoside hydrolase family 43 beta-xylosidase (Xyl43A) from the aerobic cellulolytic bacterium, Thermobifida fusca. Temperature and pH optima of 55-60 degrees C and 5.5-6, respectively, were determined. The apparent K-m value was 0.55 mM, using p-nitrophenyl xylopyranoside as substrate, and the catalytic constant (k(cat)) was 6.72 s(-1). T. fusca Xyl43A contains a catalytic module at the N terminus and an ancillary module (termed herein as Module-A) of undefined function at the C terminus. We expressed the two recombinant modules independently in Escherichia coli and examined their remaining catalytic activity and binding properties. The separation of the two Xyl43A modules caused the complete loss of enzymatic activity, whereas potent binding to xylan was fully maintained in the catalytic module and partially in the ancillary Module-A. Nondenaturing gel electrophoresis revealed a specific noncovalent coupling of the two modules, thereby restoring enzymatic activity to 66.7% (relative to the wild-type enzyme). Module-A contributes a phenylalanine residue that functions as an essential part of the active site, and the two juxtaposed modules function as a single functional entity.
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